Patent application title: qPCR array with IN SITU primer synthesis

Abstract:

Application of in situ oligonucleotide synthesis, using a maskless
photolithographic oligonucleotide synthesis apparatus or by other means,
for direct fabrication of polymerase chain reaction (PCR) primers in situ
in PCR reaction wells. The synthesized oligonucleotides contain an
enzymatically degradable linker sequence and a specific primer sequence.
The method may be used for manufacturing of quantitative PCR (qPCR)
arrays containing a plurality of independent qPCR assays while
eliminating the need for presynthesized primer libraries.

Claims:

1. A method for detecting target DNA sequences in a sample using the
polymerase chain reaction independently in a plurality of wells,
comprising:a) synthesizing oligonucleotides in situ in at least two spots
in each well of the plurality of wells, each of the oligonucleotides
comprising a cleavable linker sequence and a specific primer sequence in
a solution;b) simultaneously loading the plurality of wells with a
reaction master mix containing DNA template and polymerase chain reaction
reagents;c) sealing the wells and isolating the wells from each other;d)
heating the wells in an initial heat incubation step so that the
cleavable linker sequences are enzymatically degraded and the primer
sequences are released into solution; ande) utilizing the polymerase
chain reaction to detect whether the target DNA sequences are present.

2. The method for detecting target DNA sequences according to claim 1,
wherein the bottom surfaces of the wells are prepared with surface
chemistry enabling in situ oligonucleotide synthesis using a maskless
array synthesizer.

3. The method for detecting target DNA sequences according to claim 2,
wherein the samples are excited with a light source, and the amount of
polymerase chain reaction product in each well is determined by
monitoring fluorescent signal in the wells.

4. The method for detecting target DNA sequences according to claim 1,
wherein the cleavable linker sequences are degradable by uracil
N-glycosylase.

5. The method for detecting target DNA sequences according to claim 1,
wherein the cleavable linker sequences contain a restriction endonuclease
recognition sequence and primers are released during restriction
cleavage.

6. The method for detecting target DNA sequences according to claim 1,
wherein oligonucleotides are synthesized at three spots in each well,
wherein one spot is for a forward primer, one spot is for a reverse
primer, and one spot is for a fluorescent probe.

7. The method for detecting target DNA sequences according to claim 6,
wherein the fluorescent probe is a Taqman probe.

8. The method for detecting target DNA sequences according to claim 1,
wherein oligonucleotides are synthesized on two spots in each well, one
spot for a forward primer and one spot for a reverse primer.

9. The method for detecting target DNA sequences according to claim 1,
wherein the wells are sealed using a transparent plate.

10. A method of preparing a quantitative polymerase chain reaction array,
comprising:a) synthesizing oligonucleotides in at least two spots in each
well of the plurality of wells, each of the oligonucleotides comprising a
cleavable linker sequence and a specific primer sequence;b)
simultaneously loading the plurality of wells with a reaction master mix
containing DNA template and polymerase chain reaction reagents;c) sealing
the wells and isolating the wells from each other;d) heating the wells in
an initial heat incubation step so that the cleavable linker sequences
are enzymatically degraded and the primer sequences are released into
solution; ande) utilizing the polymerase chain reaction to amplify the
oligonucleotides to detect whether target DNA sequences are present.

[0002]DNA microarrays have become standard tools in biological and
biomedical research. They enable simultaneous quantification of thousands
of specific DNA sequences in parallel. In principle, a microarray
consists of small features (probes) of DNA spotted or by other means
attached to a flat substrate. Each probe has a unique sequence which is
complementary to a certain target DNA sequence. By hybridizing a
fluorescently labelled DNA or RNA sample onto the array, the relative
amounts of different nucleic acid sequence species in the sample may be
determined. Microarrays are used extensively for gene expression
profiling in many applications including the discovery of gene function,
drug evaluation, pathway dissection and classification of clinical
samples. They are also used for highly parallel allele discrimination,
e.g. single nucleotide polymorphism genotyping. Arrays may be produced
either by deposition of presynthesized DNA material or by in situ
oligonucleotide synthesis. The latter approach, where oligonucleotides
are assembled directly on the array surface, has several advantages. It
eliminates robotized spotting, which is a sensitive step often associated
with quality problems. The number of features per surface area can be
significantly increased. It also eliminates the need for presynthesis and
storage of large DNA libraries.

[0003]The polymerase chain reaction (PCR) is widely used for specific
detection and quantification of polynucleotides. The method uses a pair
of oligonucleotides, "primers", which specifically bind (anneal) to
specific locations on a longer DNA molecule. The region in between and
including the primers is copied several thousand fold using a cyclic
enzymatic reaction based on the use of a heat-tolerant DNA polymerase.
Applications include gene expression analysis, single nucleotide
polymorphism (SNP) genotyping and chromatin immunoprecipitation (ChIP)
studies. PCR is often used together with either a sequence non-specific
fluorescent reporter dye such as SYBR green (Wittwer C T et al.,
Biotechniques. 1997 January; 22(1):176-81) or a sequence specific
fluorescent reporter such as a taqman probe (Heid C A et al., Genome Res.
1996 October; 6(10):986-94). By monitoring the fluorescence in the sample
during the reaction, this principally simple extension of PCR provides
precise quantitative measurements (quantitative PCR, qPCR, real-time
PCR). Real-time PCR is commonly used for validation of findings
discovered using DNA microarrays and is considered to be the gold
standard for gene expression quantification.

[0004]PCR or qPCR is typically performed in plastic 96 or 384 well
microtiter plates, each reaction having a volume in the order of 5-50
μl. PCR can however be carried out in very small (nanoliter) volumes.
Miniaturization and parallelization of PCR is an area of active research
and development. One example is provided in (Dahl A et al, Biomed
Microdevices. 2007 June; 9(3):307-14) where the reactions are performed
in an array of 1024 distinct 200 nl wells in a polypropylene plate. This
type of technology has the potential to combine the sensitivity,
precision and wide dynamic range of qPCR with the parallelism of
conventional hybridization DNA microarrays. A related system is the
BioTrove OpenArray platform (U.S. Pat. No. 6,716,629), where PCR
reactions are performed in several thousand nanoliter through-holes in a
microscope slide-sized metal plate.

[0005]A major problem with the currently proposed array-format systems for
qPCR is the application of specific primers (and, where applicable,
probes) in each reaction well. For example, in a gene expression
application one is often interested in measuring the expression of a
large number of different genes in a small number of samples. Large
collections of PCR primers thus have to be synthesized and individually
applied to the miniscule reaction wells using robotized microdispensers.
This is a sensitive and inflexible solution, since the arrays cannot be
easily redesigned and each new application (biological species, type of
assay etc) requires synthesis of a new large primer library. Due to the
facts mentioned above, it is seen that a better method for manufacturing
qPCR arrays is desired.

[0006]A successful technique for manufacturing DNA microarrays is the
photolithographic method. The process begins by coating a flat substrate
with a light-sensitive chemical compound that prevents coupling between
the wafer and the first nucleotide of the DNA probe being created. The
surface is then selectively exposed to light at specific locations.
Subsequent flooding with a solution containing either adenine, thymine,
cytosine, or guanine will cause coupling to occur only in those regions
on the glass that have been deprotected through illumination. The coupled
nucleotide also bears a light-sensitive protecting group, so that the
cycle can be repeated. In this way, the microarray is built as the probes
are synthesized through repeated cycles of deprotection and coupling
until oligonucleotides of a desired length and sequence are obtained.
This system produces very high-density microarrays and is described e.g.
in U.S. Pat. No. 5,424,186 and U.S. Pat. No. 5,445,934. The method is
well established and also described in numerous articles (Fodor S et al,
Science. 251, 767-773 (1991), Jacobs, J. W. and Fodor, S. P., Trends
Biotechnol. 12(1):19-26 (1994)). Photolithographic oligonucleotide
synthesis traditionally uses lithographic masks to control the pattern of
light projected onto the array. However, instruments referred to as
maskless array synthesizers have been developed (U.S. Pat. No.
6,375,903). In these devices, the photolithographic masks are replaced by
a computer controlled micromirror device of a type similar to that used
in multimedia projectors. The light patterns projected onto the array
surface are thereby completely controlled by software and the
oligonucleotide sequences which are to be synthesized on the surface can
be changed at any time with little effort.

[0007]An alternative method for in situ oligonucleotide synthesis is the
inkjet method (see e.g., Blanchard, International Patent Publication WO
98/41531, published Sep. 24, 1998; Blanchard et al., 1996, Biosensors and
Bioelectronics 11:687-690; Blanchard, 1998, in Synthetic DNA Arrays in
Genetic Engineering, Vol. 20, J. K. Setlow, Ed., Plenum Press, New York
at pages 111-123). This is also described in U.S. Pat. No. 7,013,221 B1.
In this case, an ink jet printer head is used to apply nucleotides to
specific locations on the array surface. This enables arbitrary sequences
to be generated on the surface and provides a flexibility which is
comparable to the maskless photolithographic method.

[0008]U.S. Patent Application No 2006/0147969 relates to the use of
photolithographic preparation of high numbers of parallel sets of primers
for PCR. Methods are provided for releasing oligonucleotides from the
array surface. The only oligonucleotides that remain in position on the
array are only those used for quality control. Unlike the invention
described herein, the primers are not described to be prepared directly
in reaction wells ready to use for PCR in situ.

[0009]U.S. Patent Application No 2005/0227263 describes a method for
performing PCR amplification on a microarray. However, the patent
describes a different approach based on a protocol which results in
attachment of flanking universal primer sequences to a target DNA
sequence. After completion of this step, the target sequence can be
amplified using a pair of universal PCR primers. These primers are added
to the general reaction mixture. The invention described herein
significantly differs from this approach. No primers are added to the
reaction mixture and there is no intermediate step where flanking
universal primers are added to the target sequence. Instead, target
specific primers are synthesized in each reaction well and released into
solution using enzymatic degradation of a linker sequence.

[0010]It is therefore an object of the invention to provide an in situ
oligonucleotide synthesis method for direct fabrication of polymerase
chain reaction (PCR) primers in situ in PCR reaction wells. The present
invention describes a solution where in situ oligonucleotide synthesis is
employed for direct fabrication of PCR-primers in small volume PCR
reaction wells on a microarray. Oligonucleotides, each containing an
enzymatically degradable linker sequence and specific primer sequence,
are generated on two or more spots in each well. Before execution of the
PCR cycling program, the primer section of the oligonucleotides is
released into solution using a heat incubation step.

[0011]Other objects and advantages will be more fully apparent from the
following disclosure and appended claims.

BRIEF SUMMARY OF THE INVENTION

[0012]The present invention can be briefly summarized as a method for
parallel amplification and quantification of a plurality of target DNA
sequences in a microarray format using the polymerase chain reaction
(PCR). The method is based on the use of in situ oligonucleotide
synthesis for direct fabrication of polymerase chain reaction (PCR)
primers in situ in PCR reaction wells. Each oligonucleotide synthesized
on the well surface contains an enzymatically degradable linker sequence
and a specific primer sequence. During an initial heat incubation step,
which is simply added to the normal PCR cycling program, the linker
sequence is degraded and primers are released into solution. PCR is
subsequently performed using standard reagents and cycling conditions.
The invention may be used for manufacturing of quantitative PCR (qPCR)
arrays containing a plurality of independent qPCR assays while
eliminating the need for presynthesized primer libraries.

[0013]In a typical embodiment of the invention herein, a maskless
photolithographic oligonucleotide synthesis apparatus is used to
synthesize oligonucleotides on the bottom surfaces of small volume
reaction wells. Oligonucleotides are synthesized on two spots in each
well, one for the forward primer and one for the reverse primer. Each
nucleotide is made to contain two parts. First a linker sequence
containing U (uracil) bases is created. Synthesis continues with the
addition of a specific primer sequence. The wells are loaded with a
reaction mixture containing standard qPCR reagents (e.g. SYBR green, Taq
polymerase, dNTPs, buffer) and DNA template. In addition, the enzyme
uracil N-glycosylase (UNG) is added to the reaction mixture. Finally, the
wells are sealed using a transparent plate. By adding a heat incubation
step to the PCR cycling program (e.g. 50° C. for 10 min), primers
will be released into solution before cycling begins.

[0014]Methods and materials for testing the present invention are provided
herein. However, other related methods and materials, which are known in
the art, can also be employed. Technical terms used herein have the same
meaning as commonly accepted by those skilled in the art.

[0015]Other objects and advantages of the present invention will become
obvious to the reader and it is intended that these objects and
advantages are within the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 illustrates the general configuration of a qPCR microarray.
Each well contains two or three oligonucleotide synthesis spots according
to the invention herein.

[0017]FIGS. 2A and 2B are sectional side views of a reaction well during
synthesis of linker sequences and specific primers sequences,
respectively. The linker sequence may contain a restriction endonuclease
recognition sequence, uracil bases cleavable by UNG (uracil
N-glycosylase) or other cleavable moieties.

[0018]FIG. 3a illustrates sealing of the reaction wells using a
transparent plate.

[0019]FIG. 3b illustrates the release of PCR primers using a heat
incubation step before execution of the PCR cycling program.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

[0020]The present invention generally relates to a method for detecting
target DNA sequences using the polymerase chain reaction (PCR) in a
microarray format. It is based on the use of in situ oligonucleotide
synthesis for fabrication of PCR primer pairs directly in PCR reaction
wells. This eliminates the need for presynthesized primer libraries and
robotized dispension of primers in the reaction wells. Oligonucleotides,
each containing a cleavable linker sequence and a specific primer
sequence, are synthesized on two or more spots in each well. This leaves
the oligonucleotides firmly attached to the bottom of the wells after
synthesis. All wells are simultaneously loaded with a reaction master mix
containing DNA template and standard reagents for quantitative PCR. The
wells are then sealed and isolated from each other. During an initial
heat incubation step, which is added to the normal cycling program, the
linker is enzymatically degraded and PCR primers released into solution
prior to execution of the PCR reaction.

[0021]In situ photolithographic oligonucleotide synthesis using a maskless
array synthesizer is a well established method which is described in
detail in several patents and articles (U.S. Pat. Nos. 5,445,934 and
6,375,903). Areas on a photosensitive surface are selectively exposed to
light using a micromirror device. In areas exposed to light,
photosensitive molecules are "unprotected" to enable binding of
nucleosides containing photosensitive protective groups. By cycling
through light exposure and binding steps, thousands of unique arbitrary
sequences may be simultaneously synthesized at specific locations on the
array surface. As a consequence of the synthesis method, the final
oligonucleotides will be covalently attached to the surface.

[0022]Other in situ oligonucleotide synthesis methods may also be
employed. In another embodiment of the invention, oligonucleotides are
synthesized in situ using the ink jet method (Blanchard AP et al.
Biosensors Bioelectron 11:687-690 (1996)). In this case, nucleoside
triphosphates are selectively dispensed at specific locations using an
ink jet printer. This will also leave the finished oligonucleotides
covalently attached to the substrate.

[0023]The term "in situ oligonucleotide synthesis" used herein refers
generally to methods which synthesize oligonucleotide sequences directly
on e.g. a microarray surface. This is opposed to methods which employ a
pre-synthesized library of oligonucleotides which is later mechanically
dispensed or spotted to specific locations.

[0024]The term "PCR" as used herein refers to the polymerase chain
reaction, which is a standard method in molecular biology. It is used to
amplify (copy) a selected region of a DNA molecule. In its basic
embodiment, the method uses a pair of oligonucleotides, "primers", which
specifically bind (anneal) to specific locations of a longer DNA
molecule. The region in between and including the primers is copied
several thousand fold using a cyclic enzymatic reaction based on the use
of a heat-tolerant DNA polymerase such as Taq polymerase.

[0025]The terms "quantitative PCR" or "real-time PCR" as used herein
refers to methods where the PCR reaction is combined with fluorescence
chemistry such as SYBR green or Taqman, to enable real-time monitoring of
the amplification reaction using detection of a fluorescent light signal.
During execution of the PCR cycling program, the samples are excited
using a light source. A fluorescent signal, indicating the amount of PCR
amplification product produced, is monitored in each reaction well using
a photodetector or CCD/CMOS camera. As opposed to normal PCR, this
principally simple extension of the basic method allows precise
quantification of specific DNA sequences in a sample.

[0026]The term "template DNA" used herein refers to any DNA molecule which
can be amplified using the PCR reaction. This includes, but is not
limited to, genomic DNA and complementary DNA (cDNA) produced from RNA
using reverse transcription. The method described herein may thus be used
e.g. for parallel genotyping of single nucleotide polymorphisms (SNPs) or
quantification of messenger RNA (mRNA) expression in any organism.

[0027]The term "primer" or "primer pair" as used herein refers to short
oligonucleotides (typically 18-25 bp) which are used in PCR to select
which DNA sequence should be amplified. The size of typical
oligonucleotides is well within the boundaries of maskless
photolithographic oligonucleotide synthesis, which can be used to
synthesize oligonucleotides of 50 bp length or more.

[0028]As to a further discussion of the manner of usage and operation of
the present invention, the same should be apparent from the above
description. Accordingly, no further discussion relating to the manner of
usage and operation will be provided.

[0029]With respect to the above description then, it is to be realized
that the optimum dimensional relationships for the parts of the
invention, to include variations in size, materials, shape, form,
function and manner of operation, assembly and use, are deemed readily
apparent and obvious to one skilled in the art.

[0030]Therefore, the foregoing is considered as illustrative only of the
principles of the invention. Further, since numerous modifications and
changes will readily occur to those skilled in the art, it is not desired
to limit the invention to the exact construction and operation shown and
described, and accordingly, all suitable modifications and equivalents
may be resorted to, falling within the scope of the invention.

[0031]In one preferred embodiment, the bottom surfaces of small open wells
on a microarray are prepared with surface chemistry (linker molecules)
which enables in situ oligonucleotide synthesis using a maskless
photolithographic array synthesizer. FIG. 1 is a schematic view of the
general arrangement of such a device. The array 1 contains wells in a
tightly packed pattern. Oligonucleotides are synthesized on two spots 2
(one for each primer) in each well 3 (shown in a magnified view in FIG.
1). Oligonucleotides are synthesized in the 5'-3' direction, leaving a
free 3' end, however 3'-5' synthesis is also conceivable as both methods
are well established. First, identical linker sequences containing U
(uracil) bases are synthesized in each spot. The linker sequences enable
later release of the primers using enzymatic degradation. FIG. 2a is a
side view of a single well 4 on an array 5 during synthesis of the linker
sequences 6. Second, shown in FIG. 2b, the actual primers 7, which will
be unique in each location, are synthesized. The primer precursor
oligonucleotides are by now firmly attached to the surface and the open
wells can be loaded with standard qPCR reagents (e.g. dNTPs, hotstart taq
polymerase, SYBR green, buffer) and template in a single pipetting step
without risk of primer contamination between wells. In addition, the
enzyme uracil N-glycosylase (UNG) is added to the reaction mixture. The
wells are now sealed using a transparent glass plate 8 or similar as
previously described (see e.g. Dahl A et al, Biomed Microdevices. 2007
June; 9(3):307-14) (FIG. 3a). qPCR can now be performed as usual with the
addition of an initial 50° C. incubation step which activates the
UNG enzyme. This will result in degradation of the linker sequences,
causing primers 9 to be released into solution before execution of the
PCR reaction (FIG. 4B). A standard real-time qPCR reaction is now
performed; the samples are excited using a light source, and the amount
of PCR product produced is continuously determined by monitoring the
fluorescent signal in the wells.

Example 2

Using a Linker Containing a Restriction Endonuclease Recognition Sequence

[0032]In other embodiments, other linkers may be employed. Using a linker
sequence containing a restriction endonuclease recognition sequence,
primers may be released using restriction cleavage. In this case,
complementary oligonucleotides are annealed to the restriction linker
sequences after synthesis. Oligonucleotide synthesis in the 3'-5'
direction may be more appropriate in this case, although both directions
are conceivable. Before loading and sealing of the array, a restriction
enzyme is added to the reaction mixture. The primers are released using
an initial heat incubation step, normally at 37° C., before
execution of the PCR program. Several enzymes can be considered. An
example of a suitable enzyme is BamHI, which has good activity in PCR
buffer. Assuming synthesis in the 3'-5' direction this leaves only a
single G base in the 3' end of the primers after cleavage. Consequently,
primers will have to be designed to carry a G base in the 3' end. Since G
bases occur fairly frequently even in non-GC-rich genomic regions, this
is not a limiting factor for most applications.

Example 3

Using Probe-Based Fluorescent Detection

[0033]In yet another embodiment, oligonucleotides are synthesized on three
different locations/spots in each reaction well. In this case, a
fluorescent oligonucleotide probe such as a Taqman probe, is synthesized
in each well in addition to the two PCR primers. The probe is synthesized
according to the same principle as the primers and the same type of
linker sequence (e.g. uracil containing) will be used. Also in this case,
standard qPCR reagents can be used, and the remaining procedures will
essentially be identical to what has been described in EXAMPLE 1 or 2.